Our Tiny Allies in Toxicological Testing Part 2 – Transparent Roundworms as Alternative Models
By Aaradhya Diwan | July 10th, 2025
The field of environmental toxicology, which assesses substances to determine their impact on human and ecosystem health, is saturated with mammalian organisms and animal models whose biology is similar or can be translated to humans. Ranging from mice to rabbits to fish, studies conducted on these vertebrates are used to assess biological and environmental endpoints and set regulatory limits.
The modern push to Replace, Reduce, and Refine (3Rs) the dependency on vertebrate animal models for toxicology testing is met by the concept of New Approach Methodologies (NAMs), which are a set of 32 alternative tests validated by the OECD and ICCVAM and are employed by the EPA. In the previous blog of this series, two of these tests discussed the viability of zebrafish and their embryos as NAMs by examining their human and environmental translatability and their potential for future applications in other forms of acute toxicity testing.
However, for evaluating the toxicity of substances on the developmental and reproductive functions of biotic systems – referred to in the EPA’s Test Guidelines as NAMs for assessing effects on environmental organisms other than humans - most experimentation in the field uses pregnant rodents and rabbits, combined with skin and eye tests. In contrast, others involve exposing the animal to the chemical and subsequently observing its effects on the individual’s development before, during, and after birth, or even across multiple generations.
This blog explores the potential of Caenorhabditis elegans as a specific model and discusses its viability as a NAM for evaluating toxicity in biotic systems. Additionally, this blog demonstrates how these models can inform the detection of the negative health impacts of chemical substances.
The Worm Model
The nematode C. elegans is a powerful biological model used primarily in basic science research due to its ease of use. Scientists prefer this roundworm for four reasons: the organism is transparent, so most physical changes or phenotypic mutations are apparent and simple to observe under a microscope; they produce many offspring per individual adult per reproductive cycle; full development from embryo to adult takes 4-7 days depending on the maintenance temperature, and their cells are consistent in location and size across all worms of the same strain. These advantages make genetic modifications and molecular biology experiments relatively easy to conduct. Additionally, worms are beneficial for developmental and reproductive toxicity testing compared to mice and fish, which can take months to evaluate changes to their reproductive capabilities. Also, this nematode species is an invertebrate; therefore, it is not considered an animal under the Animal Welfare Act and other related regulations.
According to the current list of NAMs updated by the EPA in 2021, there are no alternative evaluation methods that involve C. elegans. The most relevant NAM test guidelines are OECD 207 and 222, which assess the toxicity of substances absorbed into the soil using earthworms. These closely related, opaque cousins of C. elegans are exposed to a range of test concentrations that can have at the very least, sublethal effects. Here, C. elegans can be employed more efficiently than earthworms, with a first step in evaluating soil toxicity, as they have a much quicker growth cycle (two weeks vs. one year), and their transparency is more advantageous.
So, while the worm is prevalent in the basic sciences, how can it be consistently translated to human health for toxicity testing? With zebrafish, most organ systems operate similarly to those of humans, and fish and mammals belong to the same phylum (Chordata), which means their embryonic development processes are comparable. With worms, on the other hand, the Nematoda operates according to its own set of rules for development and reproduction, mostly relying on self-fertilization and hermaphroditism instead of male-female reproductive systems. The real potential of C. elegans lies in the similarities in cellular processes, such as cell division and genetic material transfer between generations, that exist across most organisms, alongside its unique advantages described above and its consistent, well-studied neural system, which has the potential for neurotoxicity testing.
Four Nobel Prizes in Physiology or Medicine have been awarded to scientists who used C. elegans in their research. Being one of the most mapped-out organisms that humans use as a scientific model, the microscopic worm has led to the discovery of apoptosis (or cell death) and an understanding of how AIDS and degenerative diseases progress due to this process malfunctioning within the cell, and major pharmaceuticals for medical purposes have been developed stemming from another prize awarded to the discovery of gene silencing, where basic functions and protein synthesis in an organism’s body are muted or halted.
Overall, there are numerous opportunities for using C. elegans as a model for toxicity testing, thanks to their unique advantages and translational relevance to human health, as demonstrated in applications to neuroscience and developmental biology. The current obstacles to its full implementation as a New Approach Methodology lie in the consistency and reproducibility of the experiments, as there are approximately 30 variable factors in the maintenance and treatment of these microscope-transparent roundworms, including temperature, food amount, method of toxic substance administration, and method of dispersion. Should the scientific community wish to commit to using Caenorhabditis elegans as an elegant alternative for biotic systems or as a first test in a battery of experiments for human health, it would require more focus and standardization of these variables across the studies organized.
Work Cited
https://www.sciencedirect.com/science/article/abs/pii/S0887233317301510
https://pmc.ncbi.nlm.nih.gov/articles/PMC8692742/
https://pmc.ncbi.nlm.nih.gov/articles/PMC10965660/
https://doi.org/10.2307/3227041
https://www.nytimes.com/2024/10/17/science/nobel-prizes-caenorhabditis-elegans.html
https://www.iso.org/standard/71352.html
The views expressed do not necessarily reflect the official policy or position of Johns Hopkins University or Johns Hopkins Bloomberg School of Public Health.
